844
J. Org. C h e m . 1992,57,844-851
hypochlorite in a ratio of 3 1 was irradiated at 0 OC until the yellow color had disappeared and then for an additional 0.5 h. Preparative GC was used to collect the three new products. The major product (75%) was identified as 2-chloro-1,2-epoxypropane(2). 'H NMR (300 MHz, CDCl,): 6 3.10 (1H, d, J = 4.9 Hz), 2.83 (1 H, d, J = 4.9 Hz),1.89 (3 H, 8, CHJ. The other products (15 and 10%) were identified as trans- and ck-l-chloro-l,2-epoxypropane (3 and 4), respectively. 'H NMR (3) (300 MHz, CDCI,): 6 4.82 (1H, d, J = 1.0 Hz), 3.22 (1 H, dq, J = 1.0 and 5.2 Hz) 1.36 (3 H, d, J = 5.2 Hz). 'H NMFt (4) (300 MHz, CDCl3): 6 5.16 (1H, d, J = 2.8 Hz), 3.18 (1 H, dq, J = 2.8 and 5.4 Hz), 1.49 (3 H, d, J = 5.4 Hz). Preparation of erythro- and threo -3-Chloro-lf-epoxybutane. Epoxidation of 3-chloro-1-butene with m-CPBA in refluxing CHzClzgave a 1:l mixture of the erythro and threo compounds 5 and 6. Isolation of each isomer was accomplished by preparative GC. 'H NMR (5) (300 MHz, CDC13): 6 3.64 (1H, p, J = 6.7 Hz), 3.08 (1 H, m), 2.88 (1H, t, J = 4.2 Hz),2.69 (1 H, dd, J = 4.7 and 2.5 Hz),1.61 (3 H, d, J = 6.6 Hz). 13CNMR (5) (75.5 MHz, CDCl,): 6 56.8,55.5,47.2,21.8. 'H N M R (6) (300 MHz, CDC1,): 6 3.80 (1H, p, J = 6.7 Hz),3.15 (1H, m), 2.89 (1 H, dd, J = 4.7 and 4.0 Hz), 2.72 (1H, dd, J = 4.8 and 2.5 Hz), 1.55 (3 H, d, J = 6.7 Hz). 13C NMR (6) (75.5 MHz, CDCl,): 6 57.6, 55.7, 46.4, 20.6. Preparation of trans- and cis -2-Chloro-7-oxabicyclo[4.l.O]heptane. Epoxidation of 3-chlorocyclohexene with mCPBA in refluxing CHzClzgave a 9 1 mixture of compounds 7 and 8. Separation and isolation of each isomer was accomplished by preparative GC. Treatment of each isomer with an equivalent
of concd HCl gave a single isomer of 1,3-dichlorocyclohexan-2-01 (see text). 'H NMR (7) (300 MHz, CDCl,): 6 4.35 (1H, t, J = 4.8 Hz), 3.29 (1H, d, J = 4.2 Hz), 3.25 (1H, t, J = 3.4 Hz), 1.96 (3 H, m), 1.60 (2 H, m), 1.32 (1 H, m). 'W NMR (7) (75.5 MHz, CDCl,): 6 55.0, 54.9, 52.2, 28.8, 23.1, 15.1. HRMS calcd for '2C~'H~16019SC11 132.03419, found 132.03419, 'H NMR (8) (300 3.32 MHz, CDCl,): 6 4.27 (1H, ddd, J = 10.0,5.3, and 1.9 Hz), (2 H, m), 1.70 (5 H, m), 1.27 (1H, m). 13CNMR (8) (75.5 MHz, CDCl,): 6 57.4, 56.5, 55.3, 29.4, 22.3, 20.7. HRMS calcd for 1 z C ~ H ~ ' 6 0 1 132.03419, ~Cl~ found 132.03419. Kinetics. All kinetic studies were run in replicate on pairs of substrates. The two substrates, along with PhanH, an internal standard, and a solvent (benzene or cyclohexane)were mixed in an approximate ratio of 1:1:1:0.510. Aliquota were sealed in F'yrex ampules, under a reduced pressure of N2,after three freezdhaw cycles. In each case one of the ampules was reserved for analysis of starting material. The reactions were run in a temperaturecontrolled oil bath at 70 f 0.5 OC. Reaction times were varied in order to achieve 15-90% reaction of each substrate. Relative rates were determined by disappearance of starting material, as memured by integration of 'H N M R (300 MHz) or capillary GC.
Registry NO. 1, 106-89-8; 2, 5950-21-0; 3, 21947-76-2; 4, 21947-75-1; 5, 52066-40-7; 6, 52066-41-8; 7, 137940-88-6; 8, 137940.897; l-chloro-2-methoxyethane, 627-42-9;benzyl chloride, 100-44-7; cyclohexyl chloride, 542-187;neophyl chloride, 51540-2; propylene oxide, 75-56-9; 3-chloro-2-butene, 563-52-0; 3-chlorocyclohexene, 2441-97-6; triphenylstemnane, 892-20-6; triphenylstannyl, 17272-58-1.
Isolation and Structure Determination of Pentalenolactones A, B, D, and F David E. Cane,* Jae-Kyung Sohng, and Paul G. Williard Department of Chemistry, Brown University, Providence, Rhode Island 02912
Received August 9, 1991 Four new metabolites related to pentalenolactone have been isolated, pentalenolactone A (17), B (18),D (7), and F (lo), and their structures established by a combination of 'H and 13C NMR, 'H NOE, and 'H COSY spectroscopy, assisted by molecular modeling calculations. The structure and stereochemistryof pentalenolactone D phenacyl ester (21) was established by X-ray crystallography. Each of these metabolites may be important intermediates or shunt metabolites in the biosynthesis of pentalenolactone (16).
The sesquiterpene antibiotic pentalenolactone (161, which has been isolated from a variety of Streptomyces species, is a rare example of a cyclic terpenoid produced by a prokaryotic organism (Scheme I). Following the original isolation in 1957,' the structure and absolute configuration were eventually assigned in 1970 by a combination of spectroscopic and X-ray crystallographic In addition t o exhibiting a broad spectrum of activity against a wide variety of organisms, including Gram-positive and Gram-negative bacteria, pentalenolactone has been shown to block glycolysis by selective inhibition of glyceraldehyde-3-phosphatedehydrogenase from both prokaryotic (Escherichia coli, Bacillus subtilis) as well as eukaryotic sources (yeast, spinach, rabbit mus(1) Koe, B. K.; Sobin, B. A.; Celmer, W. D. Antibiot. Annu. 1967,672. English, A. R.; McBride, T. J.; Lynch, J. E. Antibiot. Annu. 1967, 682. (2) Takeuchi, S.; Ogawa, Y.; Yonehara, H. Tetrahedron Lett. 1969, 2731. (3) Martin, D. G.; Slomp, G.; Mizsak, S.; Duchamp, D. J.; Chideeter, C. G. Tetraherdron Lett. 1972,4901. Duchamp, D. J.; Chidester, C. G. Acta Crystallogr., Sect. E 1972,28, 173.
~ l e ) .Pentalenolactone ~ has also been reported to exhibit potent and specific antiviral activity? Studies in our own laboratory have shown that pentalenolactone is a timedependent, irreversible inactivator of glyceraldehyde-3phosphate dehydrogenase whose inhibitory action is due to specific reaction with all four active-site cysteines of the tetrameric enzyme? Additional studies with model thiols have suggested that the thiol residue is alkylated by ring opening at (2-10 of the epoxy lactone moiety although this has yet to be demonstrated directly for inactivation of the enzyme itself.Bb We have demonstrated the sesquiterpenoidbiosynthetic origin of pentalenolactone' and carried out extensive (4) Duszenko, M.; Mecke, D. Mol. Eiochem. Paraeitol. 1986,19,223. Duszenko, M.; Balla, H.; Mecke, D. Eiochim. Biophye. Acta 1982, 714, 344. Hartmann, S.; Neeff, J.; H e r , U.; Mecke, D. FEES Lett. 1978,93, 339. Mann, K.; Mecke, D. Nature 1979,ze2,535. Maurer, K.-H.; Pfeiffer, F.; Zehender, H.; Mecke, D. J. Bacteriol. 1983,153,930. (5) Nakagawa, A.; Tomoda, H.; Hao, M. V.; Okano, K.; Iwai, Y.; Omura; s. J. Ahtibiot. 1986, 38, 111. (6) (a) Cane, D. E.; Sohng, J.-K. Arch. Eiochem. Biophys. 1989,270, 50. (b) Sohng, J.-K. Ph.D. Dissertation, 1991, Brown University.
0022-326319211957-08444$03.00/00 1992 American Chemical Society
J. Org. Chem., Vol. 57, No. 3, 1992 846
Pentalenolactones A, B, D, and F Scheme I
D
3R-H 4 R = glucuronic acid
2
I
C
O
I-
2
H
-
oko' 6
\
\
1H 9
11
12
/
"J "
14
15
lb
t
studies of the mechanism and stereochemistry of the enPentalenolactone has been isolated from several species zymatic cyclization of farnesyl diphosphate (1)to pentaof Streptomyces, including S. chromofucus, S.griseolenene (2), the parent hydrocarbon of the pentalenolactone chromogenes,S.baarnensis, S.arenue, S. roseogrbeus,and family of metabolites.* The cyclase itself, pentalenene S. UC5319. Accompanying pentalenolactone are numerous synthase, a monomer of M R42.5 kDa, has recently been cometabolib representing possible intermediatesor shunt ~~ of the biosynthetic conversion of pentalenene purified to homogeneity and partially s e q u e n ~ e d . ~ ~metabolites Labeled pentalenene is converted to pentalenolactone by to pentalenolactone. For example, Seto et aL have isolated cultures of Streptomyces UC5319.8 pentalenolactone G (l1),lopentalenolactone H (8),11pentalenolactone 0 (19),12 pentalenolactone P (14),'2 and (7) Cane, D. E.; Roesi, T.; Tillman, A. M.; Pachlatko, J. P. J. Am. Chem. SOC.1981, 103, 1838. Cane, D. E.; Rosei, T.; Pachlatko, J. P. Tetrahedron Lett. 1979,3639. (8) (a) Cane, D. E.; Oliver, J. S.;Harrison, P. H. M.; Abell,C.; Hubbard,B. R,h e , C. T.; Lattman J. Am. Chem. SOC.1990,112,4513. (b) Cane, D. E.; Abell, C.; Tillman, A. M. Bioorg. Chem. 1984,12,312. (c) Cane, D. E.; T h a n , A. M. J. Am. Chem. Soc. 1983,105,122. (9) Cane, D. E.;Oliver, J. S.; Sohng,J.-K.; Harrison, P. H. M.; Hubbard, B. R. Unpublished work.
(10) (a) Seto, H.; Noguchi, M.; Sankawa, U.; Iitaka, J. J. Antibiot. 1984,37,816. (b) Seto, H.; Sasaki, T.; Yonehara, H.; Uzawa,J. Tetrahedron Lett. 1978,923. (11) Seto, H.; Sasaki,T.; Uzawa, J.; Takeuchi, 5.;Yonehara, H. Tetrahedron Lett. 1978,4411. (12) Seto, H.; Sasaki,T.; Yonehara, H.; Takahashi, S.;Takeuchi, M.; Kuwano, H.; Arai, M. J. Antibiot. 1984, 37, 1076.
Cane et al.
846 J. Org. Chem., Vol. 57, No. 3,1992 chart I H 0
n
7 R-H 20 R=CH3 21 R=CH&OPh
b
\
A
B
H
H
0
Ha
Figure 1. Gauche (A) and anti (B) lactone conformations of pentalenoladone metabolites.
pentalenic acid (5)" from S. chromofuscus, as well as the parent sesquiterpene hydrocarbon, pentalenene (2))from S.griseochromogene~.~~ In addition, Takahashi et aL have isolated deoxypentalenylglumn (4)14from S. omiyaensis, S. albofaciens, and S. viridifaciens. Our own group has reported the isolation and structure determination of both pentalenolactone E (S)15 and what is now termed epi-pentalenolactone F (9)t6obtained from the fermentation broth of S. UC5319. The structure of the latter metabolite was originally assigned as 10 on the basis of 'H NMR and 13CNMR spectroscopy,'& the epoxide stereochemistry being inferred by analogy to the known configuration of pentalenolactone (16) and that of the cometabolites pentalenolactones G (11) and H (8),as well as on the basis of chemical arguments." The epoxide configurational assignments were subsequently cast into doubt by further N M R studiea carried out by Matsumoto et ale'* The Hokkaido group pointed out that, based on the chemical shifts for H-12, and H-12b as well as the coupling constants, the methyl esters of observed JH-s$I-ll synthetic 9-epi-pentalenolactone H and what was then termed pentalenolactone F, but now epi-pentalenolactone F, appeared to exist in lactone conformation A (Figure 1) in which the C(5)-H(5) bond is gauche to both the C(l2)-H(12,) and C(12)-H(12b) bonds, whereas the methyl esters of pentalenolactones G and H as well as the synthetic epoxide epimer 22, now called pentalenolactone F methyl ester, existed in a second conformation (B),in which the C(5)-H(5) and C(12)-H(12b bonds have an anti relationship. When X-ray crystallagraphic analysis by Seto et al. reconfiied the correctness of the epoxide etereochemical assignment for pentalenolactone G methyl estar,'@ we carried out our own X-ray crystallographic study, leading to the now accepted revised structure 9 for naturally occurring epi-pentalenolactone F.ISb Pentalenol(13)Seto, H.; Yonehara, H. J. Antibiot. l980,33,92. (14)Takahashi, 5.;Takeuchi, M.; Ard, M. J. Antibiot. 1983,36226. (15) Cane, D. E.;h i , T.Tetrahedron Lett. 1979,2973. (16)(a)Tillman, A. M.; Cane, D. E. J. Antibiot. 1983,36,170. (b) Williard, P. G.;Sohng, J. K.; Cane, D. E. J. Antibiot. 1988,41, 130. (17)(a) OhtauIra, T.;Shirahama, H.; Mataumob, T. TetrahedronLett. 1983,24,3851.(b)Cane, D. E.; Thomas,P. J. J . Am. C k m . SOC.1984, IM,5295. (c) Danishefsky, S.;H u m , M.; Gombatz, K.; Harayama, T.; Berman, E.; Schuda, P. F.J. Am. Chem. Soc. 1979,101,7020. (18)Ohtsuka, T.; Shirahnma, H.; Matsumoto. T.Chem. Lett. 1984, 1984,1923.
a
C
O
2
R
18 R=H 24 R=CH:,
17 R=H 23 R=CHa
actone and ita structurally related cometabolitss have been the targets of numerous synthetic investigations. As reported below, we have now isolated the methyl ester of yet another metabolite, termed pentalenolactone F (lo), having the 9R epoxide configuration corresponding to the majority of naturally occurring pentalenolactones. In addition, detailed examination of the culture broths of S. UC5319 has led to the isolation of three new biogenetically related metabolites, pentalenolactones A (171,B (18))and D (7)) as described below (Scheme I and Chart I).
Rssults Treatment of the crude chloroform extracts of the acidified broth from a large-acale fermentation culture of S. UC5319 with ethereal diazomethane gave a mixture of methyl eaters which was subjected to extensive pur%cation by a combination of silica gel flash chromatography, medium and high pressure liquid chromatography, and TLC. The purified individual components were then subjected to extensive NMR analysis. In one case,the structure and stereochemistry of pentalenolactone D was further conf m e d by X-ray crystallographic analysis of the corresponding phenacyl ester 21. Pentalenolactone D. The 400-MHZ 'H NMR spectrum of pentalenolactone D methyl ester (20)showed a triplet at 6 6.83, characteristic of the conjugated olefinic (H-7) proton present in the majority of the known pentalenene or pentalenolactone metabolites. Another characteristic pair of doublets at 6 1.17 (J = 8.9 Hz, H-10, 3 H) and a quartet at 6 2.71 (J= 6.6 Hz,H-9,l H) indicated the coupling of a methyl group (H-10) and a methine proton (H-9). The high resolution mass spectrum of 20 established the elemental composition as Cl$2201. Pentalenolactone D phencyl eater (21)was m c r y d k d by the vapor diffusion method from pentane-THF (mp 158.5-159 OC).l9 The crystal of pentalenolactone D phenacyl ester grew in the monoclinic space group P21. The unit cell parameters were determined to be a = 9.267 (3) A, b = 10.634 A (3))c = 10.518 A (31, and a = y 90') B = 93.51 ( 5 ) O , by least-squares fitting to the p i t i o n e of 25 independent reflections in the range 24 O I 28 I 34O. This unit cell contained two asymmetric unita of molecular in a volume of 1034.35 (0.43) AB,which formula C23H2806 (19)Jones, P.G.Chem. Br. 1981,17,222.
J. Org. Chem., Vol. 57, No.3, 1992 847
Pentalenolactones A, B, D, and F
Table I. 'H NMR Spectral Data (CDCl,) of the Methyl Enkirn of Pentalenolactoner D (20), F (22), A (23), and B (24) and epi-PentalenolactoneF (9-Me) 20 22 9-Me 23 24 H 6 (m, J , area) H 6 (m, J , area) H 6(m,J,area) H fi(m,J,area) H 6(m,J,aren) 7 6.83 (t, 2.36. 2.32 Hz, 7 6.84 (t, 2.3. 2.4 Hz, 7 6.84 (bt, 1HI 7 6.94 (t. 2.3, 7 6.69 (t.2.2. 1 Hj
12a
4.81 (dd, 11.7, 7.0 Hz,
128
1H I 4.80 (dd, 6, 11.5 Hz,
128 16 5
3.97 (dd, 11.7, 11.7 Hz, 12a 1 H) 3.72 (e, 3 H) 16 3.36 (m, 1 H) 5
4.18 (dd, 9, 11.5 Hz, 1 H) 3.74 (e, 3 H) 3.55 (m, 1H)
16 5, 8
8
3.12 (m, 1 H)
8
2.98 (m, 1 H)
10
9
2.71 (q,6.6 Hz, 1 H)
10b
3.04 (d, 4.7 Hz,1 H)
3
10a
2.92 (d, 4.7 Hz, 1 H)
3a
1.88 (d, 12.5 Hz, 1H)
38
1.69 (d, 12.5 Hz, 1 H)
10
1.78 and 1.35 (ABq, 13.8 Hz,2 H) 1.65 (dd, 10.4, 2.84 Hz, 1HI l.&(dd, 10.4, 1.2 Hz, 1 H) 1.17 (d, 8.9 Hz,3 H)
14
1.00 (8, 3 H)
15
0.99 (e, 3 H)
la
18
1H)
1H)
12a 12b
4.77 (dd, 11.5, 2.2 Hz,1H) 4.44 (dd, 11.5, 2.2 Hz,1 H) 3.74 (e, 3 H) 3.44 (m, 2 H)
2.97 (dd, 5.2 Hz, 2 H) 1, 3 1.7, 1.43 (m, 4 H) 14, 15 1.0 (e), 0.98 (e) (6 H)
18
1.72 (dd, 9.6, 13 Hz, 1 H) la 1.43 (dd, 6.5, 13 Hz, 1 H) 14, 15 1.03 (e, 6 H)
2 Hz,1 H) 12b 4.75 (dd, 4.8, 11.8 Hz, 1 H) 12a 4.47 (dd, 4.7, 11.8 Hz, 1 H) 16 3.75 (e, 3 H) 3.5 (be,1H) 8 5
3.34 (m, 1 H)
3.21 (d, 4.6 Hz, 1 H) 10b 2.70 (d. 4.6 Hz. 1 Hj 3a 2.46 (d, 16 Hz, 1Hj 38 2.26 (d, 16 Hz, 10a
16a
12b 4.75 (dd, 5.1, 11.6 Hz, 1 H) 15b 4.71 (8, 1 H) 12a 4.35 (dd, 6.6, 11.6 Hz, 1 H) 16 3.73 (e, 3 H) 5
3.25 (m, 1H)
loa
3.18 (d, 4.64 Hz, 1 H) 1. 3.09 (m. . . 1H
8
14
1.59 (e, 3 H)
10b 2.98 (d, 4.64 Hz, 1H) 1 2.6 (m,1H)
16
1.54 (e, 3 H)
3a
1 H)
38 14
produced a calculated density of 1.23 g/cm3. A total of 1701 reflections were recorded in the range 3.5O I 28 I 4 6 O with a Nicolet R3m/E crystallographic system using the e28 scan routine and graphite-monochromatedMo Ka! radiation (A = 0.71073 A). A total of 1380 unique reflections were observed using the criterion [F, 1 3.0(F0)]. After Lorentz and polarization corrections, the structure was solved by the SHWTL 5.1 programs. All non-hydrogen a t o m were refined anisotropically. The approximate locations of all hydrogen atoms were placed in calculated positions and allowed to ride with the atom to which they are attached.20 From the X-ray structure, the measured dihedral angles in the lactone ring were H-C(5)-C(12)-HB= 48.2' and H-C(5)-C(12)-Hu = 168.1'. These dihedral angles are consistent with the observed coupling constants J H -~ ~ -12 = 7 and 11.4 Hz of pentalenolactone D phenacyl ester (21). Pentalenolactone D (7), which can be derived biogenetically by oxidation of pentalenene is conceivably a direct precursor of pentalenolactone E (6). Pentalenolactone F. Further examination of extracts of S. UC5319 led to isolation of a new metabolite, pentalenolactone F (lo), the epoxide epimer of the previously discussed epi-pentalenolactone F (9). The detailed structure of pentalenolactone F methyl ester (22) was assigned largely on the basis of N M R spectroscopic data. The high resolution mass spectrurq of 22 established the elemental composition as ClamO,. The infrared spectrum exhibited bands characteristic of a lactone (1785 cm-'), an ester (1714 cm-'),and a double bond (1620 cm-') with no hydroxyl group absorption. The 'H, 13C,and 'H COSY NMR spectra of 22 were very similar to those of epi-pentalenolactone F methyl ester (9) (Tables I and 11). The 'H NMR spectrum of 22 showed a characteristic triplet-like peak (J = 2.3 and 2.4 Hz,1H) at 6 6.84 for the H-7 conjugated olefinic proton coupled with H-8 and H-5, typical of the majority of known pentalenolactone metabolites. The 'H NMR spectrum also revealed a pair of (20)A computar-generated representation of the molecular structure of 13 can be found in the supplementary material, along with detailed X-ray crystallographic data.
2.3 &, 1 H) 4.87 (e, 1 H)
2.52 (d, 15.16 Hz, 1H) 2.24 (d, 15.16 Hz, 1 H)
1.03 (d, 6.92 Hz, 3 H)
Table 11. '42 NMR Spectral Data (CDClJ of the Methyl Esters of Pentalenolactone F (22) and epi-Pentalenolactone F (9-'Me) C 22 (ppm) 9-Me (ppm) 11 13 7 6 12 9 4 5 8 10 16 1 3 2 14 16 a-d
170.5 164.7 149.7 132.6 67.9 68.4 56.0 65.20 52.0" 51.8O 51.6 49.4 45.7 40.9 29.5' 29.1'
169.9 164.1 161.1 132.2 66.4 58.2 54.1 66.1' 66.4' 49.3 51.7 47.8
44.0 40.9
31.od 28.5d
hignments with the same letter may be interchanged.
coupled doublets centered at 6 3.04 and 2.92 (J= 4.7 Hz) resulting from the H-10 epoxide methylene protons. A pair of double doublets centered at 6 4.80 (J = 6.0,11.5 Hz,1 H) and 6 4.18 (J = 9,11.5 Hz, 1 H) corresponded to the H-12 methylene protons coupled with the H-5 proton. From these data it was clear that the new metabolite was 22, the epoxide epimer of epi-pentalenolactone F methyl ester. The stereochemistry of the epoxide moiety in 22 was unambiguously confirmed by 'H NOE experiments in combination with conformational modeling using the MACROMODEL program and an M M 2 force field?' The loweat energy conformation of 22 was calculated by energy minimization after first constraining the dihedral angles of H&
